Understanding the control of growth at the cellular level has obvious implications for the elucidation of both normal and malignant growth, and liver regeneration provides an excellent model with which to study this problem. This laboratory has previously reported experiments which demonstrate that a cytoplasmic extract of normal weanling or regenerating, but not normal, adult rat liver stimulates liver growth. This substance, which is termed hepatic stimulator substance (HSS), is organ- specific, both in vivo and in vitro, and appears to be a 12-18,000 molecular weight protein which may be acting at the level of the genome. HSS has now been purified to virtual homogeneity with a greater than 100,000 fold increase in specific activity. The present proposal consists of two parts. 1: Purification, Sequencing, Cloning and Expression of HSS. With purified HSS now available, it should be possible to sequence HSS and clone the gene for HSS. Expression of the gene for HSS with production of significant quantities of pure HSS, as well as synthesis of partial HSS sequences, will follow. cDNA probes and genomic HSS will also be prepared. Availability of purified HSS, as well as partial sequences for HSS and cDNA probes, will allow many approaches to identifying the mode of action of HSS. Hybridoma technology will also be utilized to produce monoclonal antibodies to HSS. 2. Mechanism of HSS action. Initial approaches to examining the mechanism of HSS action will focus on the early events in HSS interaction with cells. Both HTC hepatoma cells, which are very sensitive to HSS and whose response is well characterized, and normal parenchymal cells in culture will be utilized in an attempt to determine differences in the responses of the normal and malignant hepatocyte. The work will focus on the initial events which occur after exposure to HSS. Initiation events to be evaluated will include effects of HSS on ion fluxes (Na/H, Na/K pump, Ca), and effects on cellular pH. As time allows and results of the above experiments dictate, we will look at local movement of Ca between intracellular compartments, activation of the inositol triphosphate-diacylglycerol pathway and changes in cAMP levels and tyrosine kinase activation. Availability of a truly organ specific growth promotor will allow investigation of differences in growth control between normal and malignant cells. It may also prove useful clinically to stimulate liver growth in disease or trauma situations which require rapid liver growth, or as a marker of malignancy.